The class I antigen presentation pathway is critical to protect the human host against pathogens. In this pathway, nascent major histocompatibility complex (MHC) class I molecules (also called empty and peptide- free molecules) undergo a stringent maturation process within the endoplasmic reticulum (ER) that culminates in the cell-surface presentation of peptides by MHC I molecules. The recognition of peptide-filled MHC I molecules by cytotoxic T-lymphocytes (CTLs) activates the lysis of infected cells. We, and others, have shown that ER-resident proteins stabilize empty MHC I molecules in a peptide-receptive conformation. These proteins, in particular tapasin (TPN), also exert an editing function that results in high-affinity peptides being preferentially selected as part of the peptid repertoire. It is known that the conversion of MHC I molecules from a peptide-free to a peptide-filled state is accompanied by conformational changes in the antigen-binding groove. To date, the nature of these peptide-induced conformational changes remains elusive. Characterizing these conformational changes is significant because it is relevant for understanding how critical binding pockets are formed within the groove to establish tight and specific MHC I/peptide interactions. It is also relevant for understanding fundamental mechanisms of peptide loading and editing by TPN. We were the first to show that empty MHC I molecules exist as conformationally unstable and flexible species in solution. Unfortunately, such adverse properties hinder the structural characterization of these molecules, hence why the three-dimensional structure of empty MHC I molecules remains undetermined to date. In this R21 exploratory application, we will determine for the first time the x-ray crystal structure of empty MHC I molecules. For this, we propose a novel approach in which the empty molecules are stabilized by association with biologically relevant protein ligands (Aims #1 and #2). The stabilization of empty MHC I molecules by co-complexation is likely to be the only strategy to decrease the conformational heterogeneity of these molecules and allow crystallization. Upon completion of our studies, we will provide solid evidence that the MHC I peptide-receptive and peptide-filled states are structurally distinct. We will be able to explain the structural changes taking place in the groove upon peptide occupancy. Our ability to harness this knowledge will in turn advance our understanding at the structural level of how the MHC I-restricted peptide repertoire develops inside antigen-presenting cells. This is fundamental to T-cell recognition of MHC I/peptide complexes at the cell surface.